Hepatitis C virus (HCV) infection and the associated chronic hepatitis, cirrhosis and hepatocellular carcinoma are the leading cause of liver transplantation in the United States. There are approximately 4 million HCV carriers in the U.S. population. Estimates place the total number of HCV infected individuals at 170 million worldwide, representing nearly 3% of the population. During the course of HCV replication in infected individuals, the NS5a protein, a key component of the viral replicase complex, is phosphorylated by an unknown cellular kinase. This phosphorylation has been shown to also occur in cell lines carrying HCV replicons, where a link between replication efficiency and phosphorylation has been observed. The studies in this proposal are designed to identity and characterize the cellular kinase or kinases responsible for the phosphorylation of the NS5a protein of HCV by the use of conventional chromatographic separations and 2D gel electrophoresis/MALDI-TOF mass spectrometry. The effects of the observed phosphorylation of viral RNA replication will be examined using a series of replicon mutants that exhibit differing levels of NS5a phosphorylation and replication competence. The region of the NS5a protein required for the interaction with the NS5a associated kinase and the HCV replicase complex components will be identified using a battery of NS5a deletions and mutants. In addition, identification of the components of the replication complex associated with the NS5a protein and NS5a kinase will be examined and may provide some insight into the largely uncharacterized requirements for HCV viral RNA replication.